ctDNA refers to free DNA released from primary tumor cells, circulating tumor cells in the blood circulation system and necrotic or apoptotic tumor cells to the peripheral blood. Tumor refers to abnormal cell lesions, not necessarily the lumps in the body. Such lesions make cells in part of the body have uncontrolled hyperplasia, assembled into lumps. ctDNA is a biomarker with both high sensitivity and specificity. ctDNA comes from genome mutation of tumor cells, the probability of false positives is low and the half-life is short, which can accurately reflect the current situation of the tumor. With the development of high-throughput sequencing and detection technology, the importance of ctDNA detection will also promote its clinical application. High-throughput sequencing, also known as Next-generation sequencing technology (NGS), can detect ctDNA in circulating blood with high sensitivity and high specificity. By ctDNA detection, traces of tumors in blood can be detected. ctDNA carries tumor-specific mutation fragments and shows the same biological characteristics as primary tumor tissues, such that the detection of ctDNA is relatively important for tumor metastasis and prognosis.
However, after ctDNA was extracted from peripheral blood, the detection of ctDNA concentration was to detect tumor-related gene loci in the tumor samples of patients with advanced cancer, analyze the hot spot mutated genes specific to the tumor tissue by gene sequence alignment, then detect trend in changes of mutated gene loci (ctDNA) in continuous blood samples of patients by using Digital PCR technology for the subsequent application of second-generation sequencing method.
After extracting ctDNA from peripheral blood, the rapid and simple detection of ctDNA concentration is of great significance for further understanding of the tumor and treatment thereof.
Clinical studies have shown that the expression of certain genes in tumor cells is related to the sensitivity of the chemotherapeutic drugs to tumor action and the degree of drug resistance. Different tumor cells have different sensitivity to the same drug, and also same tumor cell responds differently to different drugs.
Therefore, there proposed personalized treatment. This requires the detection of multiple genes expression, currently, most of the genes need to be detected individually, the operation is complex, the convenience is poor, it is difficult to set reference genes, and it has low throughput and high cost. For PCR amplification of multiple genes in a reaction system using multiple pairs of primers, that is, multiple gene detection, it requires to repeat optimal adjustment and verification for the amplification conditions and primers. The published simultaneous detection of multiple genes generally involves almost all of the commonly used detection genes without selection, including RRM1, TOP2A, ERCC1, TYMS, TUBB3, TOP1, PTEN, HER2, DPYD and EGFR. And it is only a one-time detection, the interpretations of the detection results are not relevant, some literatures published the so-called “personalized treatment” protocol, which is mainly to detect genes in low-level, for example, if TUBB3 exhibits low-level expression, paclitaxel or vinblastine drugs are selected, and if ERCC1 is expressed at low levels, cisplatin drugs are selected, but the indication of the detected genes that show high levels of expression is not clear.